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United States Patent | 5,594,115 |
Sharma | January 14, 1997 |
Recombinant fusion proteins which comprise a biologically active polypeptide portion and metal chelating peptide are disclosed. The fusion proteins contain metal chelating peptides which have at least three and preferably six alternating histidine residues. Fusion proteins which contain metal chelating peptides that are substrates for dipeptidylpeptidase I are also disclosed. Additionally, a method of obtaining desired polypeptides by producing recombinant fusion proteins and purifying them with immobilized metal ions is disclosed. A kit for purifying desired polypeptides is also disclosed.
Inventors: | Sharma; Satish K. (Portage, MI) |
Assignee: | Pharmacia & Upjohn Company (Kalamazoo, MI) |
Appl. No.: | 365994 |
Filed: | December 29, 1994 |
Current U.S. Class: | 530/413; 435/69.7; 436/525; 530/350; 536/23.1; 536/23.4; 536/23.5 |
Intern'l Class: | G01N 033/553; C12P 021/04 |
Field of Search: | 530/350,413 436/525,808 435/172.3,69.7 536/23.4,23.1,23.5 |
4569794 | Feb., 1986 | Smith | 260/113. |
4751180 | Jun., 1988 | Cousens | 435/68. |
4782137 | Nov., 1988 | Hopp | 530/328. |
5202259 | Apr., 1993 | Goff et al. | 435/252. |
Foreign Patent Documents | |||
12709/88 | Sep., 1988 | AU | . |
0163573A1 | May., 1985 | EP | . |
0184355A3 | Jun., 1986 | EP | . |
WO88/10299 | Dec., 1988 | WO | . |
Sharma et al. 1992. Methods: A Companion to Methods in Enzymology 4:57-67. Hochuli et al. 1988 Bio/Technology 6:1321-1325. Smith, M. C. et al., "Chelating Peptide-Immobilzed Metal Ion Affinity Chromatography", J. Biol. Chem. 263(15):7211-7215, 1988. Hochuli, E., et al., "New Metal Chelate Adsorbent Selective for Proteins and Peptides Containing Neighbouring Histidine Residues", J. Chromat. 411:177-184, 1987. Ljungquist, C., et al. "Immobilization and Affinity Purification of Recombinant Proteins Using Histidine Peptide Fusions", Eur. J. Biochem. 186:563-569, 1989. Haffey, M. L. et al., "Site-Specific Cleavage of a Fusion Protein by Renin", DNA, 6(6):565-571, 1987. Hopp, T. P. et al., "A Short Polypeptide Marker Sequence Useful for Recombinant Protein Identification and Purification", Bio/Technol. 6:1204-1210, Oct. 1988. Kreil, G., "Processing of Precursors by Dipeptidylaminopeptidases: A Case of Molecular Ticketing", TIBS 15:23-26, Jan. 1990. Dalb.o slashed.ge, H. et al., "A Novel Enzymatic Method for Production of Authentic hGH from an Escherichia Coli Produced hGH-Precursor", Bio/Technol 5:161-164, Feb. 1987. Dalb.o slashed.ge, H. et al., "Cloning and Expression of an Interleukin-1.beta. Precursor and Its Conversion to Interleukin-1.beta.", FEBS 246(1,2):89-93, Mar. 1989. Dalb.o slashed.ge, H., et al., "In vivo Processing of N-Terminal Methionine in E. Coli", FEBS 266(1,2):1-3, Jun. 1990. McDonald, J. K., et al., J. Biol. Chem. 244(10):2693-2709, May 1969. |
__________________________________________________________________________ 5' AA TTC ATG CCC ATT CAC GAT CAC GAT CAC CCC TTT CAC TTA GTA ATT CAC AGC CCC ATT AGC CCT 3' 5' ATT GAG ACT GTA CCA GTA AAA TTA AAG CCA GGA ATG GAT GGC CCA AAA GTT AAA CAA TGG 3' 5' CT AAT GGG GCT GTG AAT TAC TAA GTG AAA GGG GTG ATC GTG ATC GTG AAT GGG CAT G 3' 5' CCA TTG TTT AAC TTT TGG GCC ATC CAT TCC TGG CTT TAA TTT TAC TGG TAC AGT CTC AAT AGG G 3' 5' AA TTC ATG CCC ATT CAC GAT CAC GAT CAC CCC TTT CAC TTA CTA TAC TAC AGC CCC ATT AGC CCT 3' 5' CT AAT GGG GCT GTA GTA TAG TAA GTG AAA GGG GTG ATC GTG ATC GTG AAT GGG CAT G 3' 5' AA TTC ATG CCC ATT CCC TTT CAC TTA GTA ATT CAC AGC CCC ATT AGC CCT 3' 5' CT AAT GGG GCT GTG AAT TAC TAA GTG AAA GGG AAT GGG CAT G 3' 5' AA TTC ATG CCC ATT CCC TTT CAC TTA CTA TAC TAC AGC CCC ATT AGC CCT 3' 10. 5' CT AAT GGG GCT GTA GTA TAG TAA GTG AAA GGG AAT GGG CAT G 3' __________________________________________________________________________
__________________________________________________________________________ SEQUENCE LISTING (1) GENERAL INFORMATION: (iii) NUMBER OF SEQUENCES: 20 (2) INFORMATION FOR SEQ ID NO:1: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 10 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: MetGluHisGluHisGluHisGluHisGlu 1510 (2) INFORMATION FOR SEQ ID NO:2: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 10 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: MetGluHisAspHisAspHisAspHisAsp 1510 (2) INFORMATION FOR SEQ ID NO:3: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 10 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: MetIleHisAspHisAspHisAspHisAsp 1510 (2) INFORMATION FOR SEQ ID NO:4: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 16 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: ProIleHisAspHisAspHisProPheHisLeuValIleHisSerXaa 151015 (2) INFORMATION FOR SEQ ID NO:5: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 16 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5: ProIleHisAspHisAspHisProPheHisLeuLeuTyrTyrSerXaa 151015 (2) INFORMATION FOR SEQ ID NO:6: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 11 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: ProIleProPheHisLeuValIleHisSerXaa 1510 (2) INFORMATION FOR SEQ ID NO:7: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 11 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: ProIleProPheHisLeuLeuTyrTyrSerXaa 1510 (2) INFORMATION FOR SEQ ID NO:8: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 12 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: ProIleHisAspHisAspHisProPheHisLeuXaa 1510 (2) INFORMATION FOR SEQ ID NO:9: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 65 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: unknown (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: AATTCATGCCCATTCACGATCACGATCACCCCTTTCACTTAGTAATTCACAGCCCCATTA60 GCCCT65 (2) INFORMATION FOR SEQ ID NO:10: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 60 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: unknown (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10: ATTGAGACTGTACCAGTAAAATTAAAGCCAGGAATGGATGGCCCAAAAGTTAAACAATGG60 (2) INFORMATION FOR SEQ ID NO:11: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 57 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: unknown (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11: CTAATGGGGCTGTGAATTACTAAGTGAAAGGGGTGATCGTGATCGTGAATGGGCATG57 (2) INFORMATION FOR SEQ ID NO:12: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 64 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: unknown (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12: CCATTGTTTAACTTTTGGGCCATCCATTCCTGGCTTTAATTTTACTGGTACAGTCTCAAT60 AGGG64 (2) INFORMATION FOR SEQ ID NO:13: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 65 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: unknown (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13: AATTCATGCCCATTCACGATCACGATCACCCCTTTCACTTACTATACTACAGCCCCATTA60 GCCCT65 (2) INFORMATION FOR SEQ ID NO:14: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 57 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: unknown (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14: CTAATGGGGCTGTAGTATAGTAAGTGAAAGGGGTGATCGTGATCGTGAATGGGCATG57 (2) INFORMATION FOR SEQ ID NO:15: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 50 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: unknown (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15: AATTCATGCCCATTCCCTTTCACTTAGTAATTCACAGCCCCATTAGCCCT50 (2) INFORMATION FOR SEQ ID NO:16: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 42 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: unknown (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16: CTAATGGGGCTGTGAATTACTAAGTGAAAGGGAATGGGCATG42 (2) INFORMATION FOR SEQ ID NO:17: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 50 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: unknown (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17: AATTCATGCCCATTCCCTTTCACTTACTATACTACAGCCCCATTAGCCCT50 (2) INFORMATION FOR SEQ ID NO:18: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 42 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: unknown (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18: CTAATGGGGCTGTAGTATAGTAAGTGAAAGGGAATGGGCATG42 (2) INFORMATION FOR SEQ ID NO:19: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 41 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: unknown (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19: AATTCATGCCCATTCACGATCACGATCACCCCTTTCACTTA41 (2) INFORMATION FOR SEQ ID NO:20: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 41 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: unknown (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20: GTACGGGTAAGTGCTAGTGCTAGTGGGGAAAGTGAATGATC41 __________________________________________________________________________